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Chronic pain is one of the most significant medical and scientific challenges in western society today. As well as the emotional and physical effects on the individual it proves to be a significant financial burden to society. Studies have shown that chronic pain leads to central sensitisation that is partially regulated by release of proinflammatory molecules within the CNS. Most work has concentrated on the role of the spinal cord and little is known about changes in supraspinal regions. The CFA footpad model was used to investigate the expression of inflammatory mediators in the brain in persistent inflammatory pain. Using RT-PCR, gene expression of inflammatory mediators was measured in various brain regions. Consistent with previous reports at 7 and 14 days post-injection IL1β expression was significantly elevated in the posterior of the brain (p<0.05), TNFα showed differential expression with a significant increase in the posterior ipsilateral brain region at 72 hours (p<0.05). The chemokine CXCL1 was significantly elevated at 6 hours post-injection (p<0.05) suggesting a role for this chemokine in regulation of the acute pain response. Contrary to evidence from the spinal cord, CX3CL1 and its receptor CX3CR1 were down regulated in the brain at 6 and 24 hours post-injection. Differential expression of astrocyte activation was identified by GFAP immunochemistry. Hypoxia has been implicated in neurodegeneration, a process thought to play a role in chronic pain. Here Hypoxia inducible factor (Hif1α) mRNA expression within the brain was not altered in a CFA model of peripheral inflammation. Interestingly, using Hypoxyprobe immunohistochemistry, a higher level of hypoxia was identified in non-injected controls than in CFA treated animals. This is the first evidence of differential chemokine expression in the brain in persistent inflammatory pain and the first study to suggest a potential role for differential oxygenation within the brain in this condition.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen with ability to establish a persistent infection in the host for many years. Diseases are more serious in congenitally infected infants and immunocompromised individuals such as AIDS patients and transplant recipients. It rarely causes disease among immunocompetent individuals. The envelope glycoproteins of HCMV (gB, gH, gN, gM, gL and gO) are essential for viral infectivity as they are involved in attachment and penetration of the host cell, cell to cell viral transmission and fusion of infected cells. Also, they are known to be important targets for humoral and cell mediated immune responses against the virus. The hypothesis states that HCMV genotypes are constantly mutating and evolving, then is controlled in a setting of immunocompetence, but in immunocompromised patients, congenitally infected patients or possibly within the CNS, the virus is able to evade the immune system and genotypes may evolve which are more pathogenic and/or neuroinvasive. In current study, a total of 669 CSF samples were collected from Malawian children with neurological illness that have been tested for the presence of HCMV. A comparison study population was also studied which contained 26 HCMV isolates obtained from the Manchester Royal Infirmary Clinical Virology Laboratory. The isolates were originally collected from congenitally CMV infected infants or immunocompromised children and adults showing symptoms of HCMV disease. Positive HCMV samples were screened for glycoproteins (B, H, N, L and O) and then glycotyped by restriction fragment length polymorphisms and sequencing analysis. HCMV DNA was found to be present in 46 (6.7%) out of 669 CSF samples. In 19 of these HCMV positive samples, glycoprotein genotypes (B, H, N, L and O) were identified. The predominant glycoprotein genotypes in the CSF population were as follows, gB type 3(63.2%), gH type 1(73.2%), gN type 4 (50%), gL type 4 (55.8%) and gO type 1 (60%), whilst in the comparison study population (control group) the most common glycoprotein genotypes were; gB type 3 (52.3%), gH type 1 (52%), gN type 4 (60%), gL type 4 (56%) and gO type 1(48%). No statistically significant difference was found between glycoprotein genotypes among the CSF samples and comparison study population. However, when linkage between glycoprotein types was studied and analysed differences between the two populations emerged. The particular glycoprotein linkage (gH1, gN1 and gO1) occured six times in the CSF study population with severe and fatal outcome as it occured four times with fatal outcome, and in two other patients, one was HIV positive and one with sequelae.Novel linkages between gO and gN genotypes were also found (gN3b/gO3), (gN4a/gO4), (gN4b/gO2b), (gN4b/gO3), (gN4d/gO1c), (gN1/gO1c), and (gN4c/gO4). The (gN1/gO1c) combination was occurred three times, but only in CSF study population and was associated with fatal outcomes, two patients died and one was HIV positive patients.RFLP assay was found to have limited efficacy in identification of HCMV genotypes, as discrepancies in genotype results between RFLP and sequencing were found in all glycoprotein genotypes. Thus, sequencing analysis was more accurate and recommended for identification of the HCMV genotypes.In conclusion, investigation of different glycoprotein genotypes in CSF samples has shown no significant correlation between single glycoprotein and disease outcome. Recombination between HCMV strains may lead to progression of disease. However, studying a large group of CSF samples positive for HCMV from immunocompromised patients may clarify the correlation between glycoprotein types and neurological outcome, and lead to improved strategies for treatment and prevention of HCMV associated disease.

Studiens syfte var dels att undersöka om det fanns skillnader iförskrivning av centralstimulantia hos barn som kunde kopplas tillföräldrarnas utbildningsnivå och dels att undersöka om det fannsskillnader i förskrivning av centralstimulantia vid nydiagnosticeradADHD som kunde kopplas till föräldrarnas utbildningsnivå. Uppgifterom studiepopulationen hämtades som aggregerad data frånläkemedelsregistret, patientregistret och utbildningsregistret.Deltagarantal för första forskningsfrågan var 37959 personer, varav25385 pojkar och 12574 flickor. Den andra forskningsfråganomfattade 9443 personer varav 5931 pojkar och 3512 flickor.Åldrarna på deltagarna var 5-24 år. Resultaten från analyser av denförsta forskningsfrågan visade att barn vars föräldrar hadegymnasium som högsta utbildning var den grupp som hade störstandel behandlade. I den andra forskningsfrågan sågs inga skillnadermellan föräldrarnas utbildningsnivåer och andelen behandlade vidnydiagnosticerad ADHD. / This study examined some factors that can influence which childrenthat medicate with Central Nervous System (CNS) drug. Are there alink between differences in CNS drug use for children with ADHDconnected to parents’ educational level. The research questions of thisstudy were: 1) What are the impacts of parents’ education level onCNS drug use by children with ADHD? and 2) Are there differencesbetween these group of children and children newly diagnosed withADHD in this regard? Aggregated data for this study was collectedfrom The national patient register, Pharmaceutical register, andEducation register. Number of participants in the first group was37959 and the second group consisted of 9443 children. The resultsshowed that children whose parents had Swedish gymnasium as theirhighest education were the ones with the highest treatment proportion,but there were no differences in time of treatment considering theparental education level.

Exosomes are small lipid membrane bound vesicles formed as part of the endosomal pathway and released into the extracellular space following fusion of late endosomes with the plasma membrane. Exosomes have been shown to have a variety of biological roles and may represent a novel source of disease biomarkers. The objectives of this project were to develop a panel of techniques for identifying exosomes in human urine then establish an in vitro model to determine whether exosomes change with cellular activation. We then used the techniques developed with human urine to determine whether human cerebrospinal fluid (CSF) contains exosomes and applied a mass spectrometry based approach to characterise the exosomal proteome. We used western blot for three exosomal markers (tsg101, CD24 and flotillin- 1), isopycnic centrifugation on a sucrose density gradient and direct visualisation using transmission electron microscopy (TEM) to verify the presence of exosomes. Using GeLC-MS/MS, 88 proteins were identified in the urinary exosomes. Several of these proteins could be linked to diseases and specific sections of the nephron. A murine cortical collecting duct cell line was used to model exosome release into the urine. Firstly, exosome release was verified using the approach developed in the urine. Stimulation of the cells with desmopressin caused an increase in the presence of aquaporin 2 in the exosomes. This increase reflected a similar change in the cells and occurred over a similar time course. This supports the hypothesis that the exosomes reflect the state of the kidney cells. In contrast, stimulation with cisplatin did not alter the presence of Fetuin-A, a proposed biomarker of cisplatin-induced acute kidney injury, in exosomes and this was consistent with no change in Fetuin- A expression in the cells. The released exosomes may act as mediators of communication to other cells. Following incubation of mCCD cells with AQP2 containing exosomes AQP2 in the cell lysate was increased indicating interaction between the cells and exosomes and potentially internalisation. Exosomes have been shown to be released by neuronal cells in vitro. We identified exosomes in the CSF of humans using western blot for known exosomal markers, density determination and direct visualisation with TEM and Immuno-TEM using an antibody specific for the exosomal marker flotillin-1. Label-free quantitative mass spectrometry was used to compare multiple CSF samples. On a whole protein analysis 86% of the proteins identified varied by less than 2-fold in comparison to the average across samples. On a tryptic peptide analysis 75% of the peptides identified varied by less than 2-fold in comparison with the average across samples. We have demonstrated exosomes are present in urine, CSF and mCCD cell conditioned media. In the mCCD cell derived exosomes we have demonstrated that following stimulation the proteome of the exosomes changes and that this change reflects the change seen in the cells. For the urinary and CSF exosomes we have characterised their proteomes using GeLC-MS/MS. These findings are consistent with the hypothesis that exosomes are a rich source of information, including biomarkers, on their cells of origin.

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